single respiratory collection system, starplex s

M-52
SINGLE RESPIRATORY COLLECTION SYSTEM, STARPLEX S-160 NASO, FOR MULTIPLE INVESTIGATIONS
R.WHEELER*, T. HAYES, D. HATHAWAY, D. JASKOT, J. KRAHN, S. MILBURN, A. TIROLESE, R. LANNIGAN
Virology Department, London Laboratory Services Group, London, Ontario, Canada
Abstract
Objective: The usual route for bacterial and viral pathogen identification relies on the collection of separate patient samples. Depending on
the clinical symptoms there is an opportunity to lessen the invasiveness
of the sample collection process. In the early stages of a Bordetella
pertussis infection, the symptoms resemble those of a common cold
and if the patient is in the first two to three years of life, multiple viral
pathogens may also be the causative agent. In this facility the only diagnostic test utilized to identify Whooping cough is a Real-Time PCR
assay and is run in conjunction with viral DFA and culture for routine
samples, and may also include multiplex PCR detection of viruses. A
single sample collection and transport system (S-160 NASO) has been
validated to give optimal patient results.
Method: Nasopharyngeal swabs are collected for the investigation of
B. pertussis alone, a panel of viruses or a combination of the two, depending on the presentation. A real-time assay targeting the IS 481
region of B. pertussis is utilized to diagnose Whooping cough, while
DFA testing with culture back-up is utilized to identify eight major viral
pathogens on a routine basis. Multiplex viral PCR investigation is performed for institution outbreaks and special requests where standard
tests do not identify a pathogen.
Results: Samples collected in the transport media showed no inhibitory effects on the PCR assays either for the real-time B. pertussis
assay or in the viral multiplex assay developed by Luminex Molecular
Diagnostics. A total of 140 samples were received over a period of 8
months for identification of B. pertussis alone or in combination with
viral investigation. In the patient population ranging from 4–17 years of
age, 24% of the samples were for B. pertussis alone. In the <= 3 years
age group B. pertussis was ordered in conjunction with viral investigation (76%) with the majority (85%) of those being under 2 years of age.
Despite the literature reflecting a rise in detection of whooping cough
(for multiple reasons) this facility showed only a 2% positivity rate, while
viral pathogens were detected at a rate of 57%. Of the viruses identified 43% were Respiratory Syncytial Virus (RSV), 9% were Parainfluenza and 3% each for Influenza B and Metapneumovirus. Dual viral
infections were also detected at a rate of 17 % while viral and bacterial
(B. pertussis and RSV) infections at 1%. These percentages fluctuate
year to year based on circulating pockets of infectious agents.
Conclusions: Sample collection can be a challenge and sub-optimal
samples can result in anything from useless to misleading information.
When multiple diagnostic tests can be run reliably on a single sample
the outcome will be beneficial to the laboratory, care provider and patient. The S-160 NASO has proven to be a reliable component of the
diagnostic process. A collection procedure that is well tolerated leads
to a high quality sample for a number of testing algorithms that may include direct antigen testing, culture amplification (selected pathogens)
and molecular based assays without any demonstrated interference.
Introduction
While the testing policy and process for Bordetella pertussis was be-
ing investigated, cited literature indicated that direct testing and culture
lacked the required sensitivity. As a result we decided to rely on a molecular based assay for identification of this reportable disease. Due to
its presentation concurrent testing for viral pathogens is often requested,
requiring a separate swab. Simultaneously the collection systems for
viral respiratory illnesses were in transition due to the Post-SARS experience and need to achieve high quality specimens that were easily collected. The result was the implementation of a single Starplex Scientific
S-160 NASO for all viral and B. pertussis diagnostics.
Results
One of the main advantages of switching to a single recovery sample
collection system for multiple investigations has been the reduced invasiveness and higher comfort level to the patient. The move away
from aspirate collection has improved Infection Control measures in the
hospital and almost eliminated the labs reception of leaking samples.
It has improved upon cellular material recovery with the elimination of
samples labeled as NP aspirates, but resembling a sputum collection.
Conventional Swab
Flocked Swab
DFA
Culture Confirmation
A published protocol for Real-Time PCR detection1 was adopted by the
lab replacing a previous published protocol utilizing conventional PCR
and gel detection. The assay utilizes the Roche LightCycler Fast Start
DNA Master PLUS Hybridization Probe kit and primers and probes are
directed at the IS 481 region of B. pertussis. Extractions are performed
using the Roche MagNA Pure Compact instrument and Total Nucleic
Acid Isolation Kit reagents.
When institutional outbreaks go undiagnosed or in special cases cleared
by the Medical Microbiologist, the xTAGTM RVP from Luminex Molecular
Diagnostics is employed. Extraction procedures are the same for B. pertussis and this assay normally requires 6.5 hours of technologist time to
complete.
Negatives
Viral Agent
Positives
Total
B. pertussis
Requests
B. pertussis
Positives
34
2
N/A
36
B. pertussis +
Viral Requests
44
1*
59*
104
TOTAL
78
3
59
140
• Single patient sample(*) dual infection with B. pertussis and RSV
• Note: Recent single respiratory sample received tested positive for B. pertussis, Influenza A and RSV (April 2008)
Molecular analysis with xTAGTM RVP from Luminex Molecular Diagnostics was the primary method for detection of dual infections. PCR inhibition did not present a problem with the use of this collection system in
either molecular amplification assay. All of the dual infections that did
not involve Entero/Rhino viruses were confirmed by culture isolation.
Current sample protocols do not include extended incubation times or
the use of screening antibodies to detect members of the Picornaviridae family. As expected in the younger patient population the predominant virus identified was Respiratory Syncytial Virus (43%) and with
the help of RVP almost equal numbers of Type A and B were detected.
The remainder of the viruses identified in our geographic area included
Parainfluenza (Type 1 and 3), Influenza B and Metapneumovirus.
Conclusion
on.ca/lab/MICRO/virology/virology_videos.htm
Standardized published sample handling procedures are utilized for producing DFA slide material for rapid testing, followed by a culture confirmation backup of all samples, possibly excluding institutional outbreaks.
The laboratory currently examines both direct smears and tissue culture
for Influenza A/B, RSV, Parainfluenza 1/2/3, Adeno and Metapneumovirus. Shell vial culture from Diagnostic Hybrids (R-Mix) and antibody
reagent material from Chemicon and DHI are used in the viral identification.
[email protected]
Respiratory Samples Received: November 2006 – June 2007
Materials and Methods
Respiratory sample collection along with the extent of investigation falls
to the clinical teams in each area of the hospitals and long-term care
facilities. After the emergence of the SARS virus, steps were taken to
improve infection control practices when patients exhibiting respiratory
symptoms were examined. Our facilities adopted the use of swab collection systems for both children and adults, replacing the often used
aspirates. On-line educational material was made available for all staff
including a training video for proper collection technique. http://www.lhsc.
A Joint Venture between London Health Sciences Centre and
St. Joseph’s Health Care Centre, London, Ontario, Canada
RT - PCR
Although B. pertussis has seen resurgence in the last few years world
wide, the prevalence in this institution remains quite low. The ruling out
of this respiratory agent along with similarly presenting viral etiologies
does continue to be the focus of the clinical units throughout our Inpatient, Out-patient and long-term care areas.
Providing a patient with the least taxing hospital encounter coupled with
useful diagnostic information should be the goal of all health care facilities. Due to the testing algorithm of this laboratory we are allowed to
take advantage of innovative products in order to provide a high quality
service. The S-160 NASO has enabled us to provide rapid turn-aroundtime on high quality samples and supply clinically relevant and reliable
diagnostic information. A majority of sample processed are collected in
Emergency departments where clinical decisions are being made related to severity of disease and admission to the facility. The ability to
utilize a single patient sample for a series of clinical investigations has
proven to be of great benefit to the testing lab and has also contributed
to a higher level of safety for health care professionals that are involved
in patient contact.
Reference
1. Udo et. al. Real-Time PCR Assay Targeting IS 481 of Bordetella
pertussis and Molecular Basis for Detecting Bordetella holmesii. JCM,
Vol. 39, No.5, pgs 1963-1965.